The objective of the proposed research is to understand the molecular and biophysical mechanisms of ion permeation and gating of inward rectifier K (Kir) channels. Kir channels are abundant in brain, and are crucial for the maintenance of the resting membrane potential and for neuronal excitability. A better understanding of he mechanisms of Kir channel gating and permeation will help us understand how they function in normal brain and in neurological disorders such as epilepsy and stroke. The recent crystal structure of KcsA channel has provided a structural basis for K channel permeation and gating. Here we propose to use a novel method of unnatural amino acid mutagenesis to change the carbonyl oxygens lining the selectivity filter from amide to ester carbonyls. These mutations will significantly alter the physical chemical properties of the carbonyls and their interactions with permeant ions, while leaving the overall backbone structure intact. The specific aims of this proposal are: (1) To study the interactions between permeant ions and the backbone carbonyls. We will exam in he effect of the backbone mutations on ion selectivity, Ba2+ block, Cs+ block and K+ binding. (2) To test the hypothesis that dynamic conformational changes of the selectivity filter directly contribute to the gating of Kir2.1 channels. We will examine the effect of the backbone mutations on single-channel kinetics and subconductance levels with either K+ or Tl + as the permeant ion.